SARS spread to over thirty countries in 2003. There were more than 8,000 individuals infected and 800 lives were lost. A novel SARS-CoV was subsequently identified as the etiological agent of SARS (1-3), and it was further confirmed that the virus caused a similar disease in cynomolgus macaques (4). Although SARS appears to have been successfully contained, re-emergence of this life threatening disease remains a significant possibility. There have been three laboratory-acquired and four community-acquired SARS cases were recently reported in Singapore, Taiwan and China (40). Therefore, effective vaccines or antiviral drugs against this disease are urgently needed.
SARS-CoV-like viruses have been isolated from and characterized in small mammals such as civet cats and raccoon dogs, implying that these animals may be the source of SARS (5). Important factors associated with the emergence of novel infectious diseases from animal sources include extensive exposure and rapid virus evolution (6). Phylogenetic analysis has revealed that although human SARS-CoV and animal SARS CoV-like viruses are related to the three groups of the previously found coronaviruses, they are different enough to make up their own, the fourth group, which may be a big family in wildlife. Increasing consumer demands for wild/farmed animals in Guangdong, China in the past 15 years has provided an incubator to facilitate interspecies virus transmission from wild/farmed animals to domestic animals and humans. The mutation rate will increase in interspecies transmitted viruses due to novel selection pressure in the new hosts.
In SARS-CoV infection, the spike protein (“S protein”) recognizes and binds to host cell receptors, and the conformational changes induced in the S protein would then facilitate the fusion between the viral envelope and host cell membranes. Previous studies have clearly identified that there are significant sequence variations in the region encoding the S protein, with twelve notable amino acid substitution changes (5). These substitutions may hold the key to understanding why and how the virus crossed the species barrier from animals to humans in the recent outbreak. The rapid mutation of these sites was further elucidated in a recent study for SARS surveillance. By comparing animal SARS-CoV-like viruses isolated in May 2003 (5) and those isolated after October 2003 (unpublished data), further variations in these sites are identified, which are completely identical to the human SARS-CoV isolated from a patient in December 2003 in Guangzhou (7) (Table 1). The rapid mutations in these sides suggest that at least some of these mutations would play a crucial role in viral transmission across the species barrier from animals to humans. It is hypothesized that agents that interfered with, or bound competitively with these protein domains would be able to inhibit SARS-CoV infections by disrupting the function of the S protein. To test this hypothesis, ten peptides that spanned these variable regions were synthesized and their antiviral effects in a cell culture system were investigated.
TABLE 1Amino Acid Variation of S Protein Between Animals and Human SARS-CoVP1P2P3P4P5P6P7P8P9P10SamplingViruses2272392613604975586077017437548941163Animal (May 2003) (5)SZ1KLKSKIPLAVAESZ3KLKSKFPLAVAESZ16KLKSKIPLAVAEAnimal (October 2003)*Hc/SZ/266/03NSTSNFSSRATEAnimal (November 2003)*Hc/SZ/DM1/03NSTSNFSSRATEAnimal (December 2003)*Hc/GZ/32/03NSTSRFSSRATEHc/GZ/81/03NSTSNFSSRATEHc/SZ/61/03KSTSRFSSRATEHc/SZ/79/03NSTSRFSSRATECFb/SZ/94/03NSTSRFSSRATEHuman (December 2003) (7)Hu/Gz/1/04NSTSNFSSRATEHuman (February 2003) (13)GZ01NLTFNFSSTATKGZ43NLTFNFSSTATKGZ50NSTFNFSSTATKGZ60NLTFNFSSTATK*Unpublished data.Abbreviations used:Human (“Hu”);Himalayan civet (“Hc”);Chinese ferret-badger (“CFb”),Guangzhou (“GZ”); andShenzhen (“SZ”).P1-P10 are SEQ ID NOs.: 1-10, respectively.
Antiviral peptides targeting HIV-1 (8, 9), gp40 of feline immunodeficiency virus (FIV) (10), and the coiled-coil domain of human T-cell leukemia virus type-1 (HTLV-1) (11) have been demonstrated to be effective inhibitors of these viral infections, with potential therapeutic value in the treatment of the viral diseases. The inhibitory effects of these synthetic peptides were mediated by blocking the interaction of viral proteins with their cellular receptors, or alternatively, by preventing membrane fusion. Based on these findings, a recent study has demonstrated that a peptide targeting the heptad repeat 2 region of the SARS-CoV S protein inhibits virus infection in the micromolar range (12).
In this invention, peptides which target four regions of the S protein were synthesized and identified to effectively inhibit SARS CoV infection in a monkey kidney (FRhK-4) cell line. Synergistic antiviral effects were observed when cells were treated with combinations of two or three of these peptides prior to infection. 3D modeling indicated that three of the antiviral peptides map to subunit interfaces putatively crucial for the correct assembly of the trimeric peplomer. The results suggest a novel inhibitory mechanism distinct from the previously reported anti-SARS-CoV peptide, which disrupted the heptad repeat 1-heptad repeat 2 (“HR1-HR2”) interaction.
Definitions
“Peplomers” described herein means layers of protein which surround the capsid in animal viruses with tubular nucleocapsids. The envelope has an inner layer of lipids and virus specified proteins also called membrane or matrix proteins. The outer layer has one or more types of morphological subunits called peplomers which project from the viral envelope; this layer always is composed of glycoproteins.
“Subject” shall mean any animal, such as a mammal or a bird, including, without limitation, a cow, a horse, a sheep, a pig, a dog, a cat, a rodent such as a mouse or rat, a turkey, a chicken and a primate. In the preferred embodiment, the subject is a human being.
“Pharmaceutically acceptable carrier” shall mean any of the various vehicles or carriers known to those skilled in the art. For example, pharmaceutically acceptable carrier includes, but is not limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.
Peptides described herein are represented by “one-letter symbols” for amino acid residues as follows:
AAlaAlanineRArgArginineNAsnAsparagineDAspAspartic acidBAsxAsn or AspCCysCysteineQGlnGlutamineEGluGlutamic acidZGlxGln or GluGGlyGlycineHHisHistidineIIleIsoleucineLLeuLeucineKLysLysineMMetMethionineFPhePhenylalaninePProProlineSSerSerineTThrThreonineWTrpTryptophanYTyrTyrosineVValValine